Process Biochemistry 42 (2007) 279–284
www.elsevier.com/locate/procbio
Short communication
Batch and continuous fermentative production of hydrogen with
anaerobic sludge entrapped in a composite polymeric matrix
Ken-Jer Wu a,b, Jo-Shu Chang b,*
b
a
Department of Biochemical Engineering, Kao Yuan University, Kaohsiung 821, Taiwan
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
Received 19 December 2005; received in revised form 22 June 2006; accepted 13 July 2006
Abstract
Cell immobilization techniques were adopted to biohydrogen production using immobilized anaerobic sludge as the seed culture. Sucrosebased synthetic wastewater was converted to H2 using batch and continuous cultures. A novel composite polymeric material comprising
polymethyl methacrylate (PMMA), collagen, and activated carbon was used to entrap biomass for H2 production. Using the PMMA immobilized
cells, the favorable conditions for batch H2 fermentation were 35 8C, pH 6.0, and an 20 g COD l1 of sucrose, giving a H2 production rate of
238 ml h1 l1 and a H2 yield of 2.25 mol H2 mol sucrose1. Under these optimal conditions, continuous H2 fermentation was conducted at a
hydraulic retention time (HRT) of 4–8 h, giving the best H2-producing rate of 1.8 l h1 l1 (over seven-fold of the best batch result) at a HRT of 6 h
and a H2 yield of 2.0 mol H2 mol sucrose1. The sucrose conversion was essentially over 90% in all runs. The biogas consisted of only H2 and CO2.
The major soluble metabolites were butyric acid, acetic acid, and 2,3-butandiol, while a small amount of ethanol also detected. The PMMAimmobilized-cell system developed in this work seems to be a promising H2-producing process due to the high stability in continuous operations
and the capability of achieving a competitively high H2 production rate under a relatively low organic loading rate.
# 2006 Elsevier Ltd. All rights reserved.
Keywords: Anaerobic sludge; Biohydrogen production; Continuous culture; Immobilized cell; Polymethyl methacrylate
1. Introduction
Hydrogen has been recognized as an ideal energy carrier of
the future because it is clean, recyclable, and efficient [1,2].
Being a country importing over 95% of its energy demand,
Taiwan has sensed the importance of this new energy carrier
and has been devoted to the development of hydrogen energy
technology. Production of H2 is one of the vital components in
H2 energy platform. Biological production of H2 provides a
feasible means for the sustainable supply of H2 with low
pollution and high efficiency, thereby being considered a
promising way of producing H2 [1,2]. Fermentative H2
production can be achieved by dark fermentation (with
obligate or facultative anaerobes) or by photo fermentation
(with photoheterotrophic bacteria) [1,2]. Among them, dark
fermentation normally achieves a much higher H2 production
* Corresponding author. Tel.: +886 6 2757575x62651;
fax: +886 6 2357146/2344496.
E-mail address: [email protected] (J.-S. Chang).
1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2006.07.021
rate and is considered more applicable for simultaneous waste
reduction and H2 generation [1].
Cell immobilization technology has been successfully
applied to fermentation and enzymatic transformation [3].
However, the technology has not been widely adopted to H2
production through dark fermentation, whereas there were
some examples describing using immobilized cells for
phototrophic H2 production [4,5]. Our recent work had
developed several immobilized-cell systems for dark H2
fermentation [6,7]. Those cell-entrapment type immobilized
cells were effective in H2 production on batch mode but most
of them were either infeasible in continuous operations (e.g.,
calcium alginate (CA)-based and polyurethane (PU) immobilized cells [6]) or have not yet been tested in continuous
operations (e.g., immobilized cells created by ethylene-vinyl
acetate (EVA) copolymer [7]). In this work, we attempted to
create more stable and reliable immobilized cells able to be
used on continuous operations. In addition, the effect of the
medium and environmental factors was investigated to
identify favorable conditions for H2 production with the
immobilized-cell system. This work is to our knowledge one
280
K.-J Wu, J.-S Chang / Process Biochemistry 42 (2007) 279–284
of the early attempts in using immobilized bacterial microflora for continuous dark H2 production [8]. The outcome of
this work is expected to provide useful information for future
development of commercial viable bioprocesses for H2
production.
from the culture was measured with a gas meter (Type TG1; Ritter Inc.,
Germany) with a measuring limit of 10 ml. The gas volumes presented in this
work were standardized to 25 8C and 760 mmHg. The time-course H2 evolution
data were simulated with modified Gompertz equation (Eq. (1)) [10] to
determine the H2 production potential (Hmax), maximum H2 production rate
(Rmax), and the lag time (l):
2. Materials and methods
Rmax;H2 e
H ¼ H max exp exp
ðl tÞ þ 1
H max
2.1. Hydrogen-producing sludge and fermentation medium
The seed sludge was collected from the final sedimentation tank of a
municipal activated-sludge-based wastewater treatment plant located in central
Taiwan. Prior to use, the sludge was subjected to acidic pretreatment [9] and was
then acclimated at 35 8C in a continuous-flow reactor operated at a HRT of 6–
12 h to enrich its H2 producing activity. The medium for cell growth and H2
production contained 20 g COD sucrose l1 (adjustable) as the sole carbon
source as well as sufficient amounts of inorganic salts [9].
2.2. Immobilization of anaerobic sludge
Fifty milliliters (ca. 0.15 g VSS) of acclimated H2-producing sludge was
mixed with 25 g of polymethyl methacrylate (PMMA). Supplemental materials (15 g collagen and 10 g activated carbon) were added to modify the
physical properties (e.g., density, pore size, mechanical strength, etc.) of
the immobilized cells. The core matrix, supplemental materials, and sludge
were mixed thoroughly at 45 8C. The colloid mixture was transferred to a
syringe, and was then extruded to form disc-like beads with a diameter of
0.5 cm and a volume of ca. 0.25 cm3. No coagulation/curing solution was used
after bead extrusion.
2.3. Batch H2 fermentation
Twenty-five grams of PMMA-immobilized beads (containing ca. 0.15 g
biomass) was inoculated into a 250-ml serum vial containing 100 ml of the
aforementioned medium. After inoculation, the vial was sparged thoroughly
with argon gas to create an anaerobic condition. The batch operations were
conducted at different sucrose concentration (5–30 g COD l1), pH (5.5–7.0),
and temperature (30–40 8C) under a fixed agitation rate of 100 rpm. The
composition of gaseous (mainly H2 and CO2) and soluble products (volatile
fatty acids and alcohols) was monitored with respect to time. The gas produced
(1)
The volumetric H2 production rate and H2 yield were used as major
performance indexes assessing the performance of H2 production. The volumetric H2 production rate was determined based on the kinetic constant
estimated from modified Gompertz equation (Eq. (1)). The yield was defined
as mol of H2 formed per mol of sucrose consumed.
2.4. Continuous H2 fermentation
A 2.5-l jar fermentor was used for continuous H2 production using the
PMMA-immobilized cells. About 100 g of immobilized beads (containing
ca. 0.6 g biomass) were added into 1 l of medium containing 20 g COD l1
of sucrose. The continuous fermentation was operated at 35 8C, pH 6.0,
200 rpm agitation, and a hydraulic retention time (HRT) of 4–8 h. The
continuous culture was started up at a HRT of 8 h. While a stable operation
was reached, the HRT was progressively decreased from 8 to 6 h, and then
finally to 4 h. A gas meter was connected to the gas effluent of the
fermentor to measure the amount of biogas produced. Samples were taken
from at designated time intervals to detect the gas products and soluble
metabolites.
2.5. Analytical methods
The gas products were analyzed by gas chromatography (GC) using a
thermal conductivity detector (TCD). The volatile fatty acids and ethanol
were also detected by GC using a flame ionization detector (FID). The
details for GC analysis were described in our recent report [9,11,12].
Standard methods [13] were used to measure the carbohydrate concentration
in the effluent and to determine the biomass concentration (in terms of
volatile suspended solid; VSS) of the sludge samples. The biomass content
in immobilized beads was examined at the end of experiment by mechanical
disruption of the beads, followed by similar weighing procedures for VSS
measurement.
Table 1
H2 production performance and estimated kinetic parameters with modified Gompertz equation (Eq. (1)) in batch fermentation using PMMA immobilized cells under
different combinations of temperature and initial pH
Run
1
2
3
4
5
6
7
8
9
10
11
12
Temperature
(8C)
Initial
pHa
H2
contentb (%)
H2 production
rateb,c (ml h1 l1)
H2 yieldb
(mol H2 mol sucrose1)
30
30
30
30
35
35
35
35
40
40
40
40
5.5
6.0
6.5
7.0
5.5
6.0
6.5
7.0
5.5
6.0
6.5
7.0
48 2
47 1
27 0
27 1
49 3
43 1
39 1
45 0
50 2
39 0
40 1
40 2
28 2
25 1
33 3
49 2
95 7
238 10
109 4
136 3
129 9
104 8
60 4
44 1
0.31 0.02
0.39 0.03
0.41 0.04
0.30 0.01
1.21 0.07
2.25 0.12
1.42 0.09
1.58 0.17
0.91 0.07
0.80 0.04
1.53 0.11
1.41 0.08
Initial sucrose concentration = 20 g COD l1.
a
The final pH was within the range of 4.5–4.8.
b
The data are the mean values of duplicate tests (the ‘‘’’ denotes standard deviation of duplicates).
c
Maximum volumetric H2 production rate.
Model simulation
Total H2 evolution,
Hmax (ml)
Rmax
(ml h1)
l (h)
R2
34
49
51
36
135
252
158
181
109
96
171
165
2.8
2.5
3.3
4.9
9.5
23.8
10.9
13.6
12.9
10.4
6.0
4.4
8.2
8.5
8.5
7.8
7.7
7.8
8.2
8.1
7.8
7.7
7.7
8.1
0.970
0.998
0.990
0.964
0.986
0.995
0.995
0.998
0.963
0.950
0.964
0.942
K.-J Wu, J.-S Chang / Process Biochemistry 42 (2007) 279–284
281
3. Results and discussion
3.1. Batch H2 production with PMMA-immobilized cells
To characterize the H2-producing performance of the
PMMA immobilized cells, three critical influential factors
(temperature, pH, and carbon substrate concentration) on dark
H2 fermentation were examined [14]. The kinetic data
obtained from batch cultures conducted at various pH and
temperatures were simulated by modified Gompertz equation
(Eq. (1)). Table 1 shows that the best H2 production rate
(Rmax), total H2 evolution (Hmax), and H2 yield (Y H2 ) occurred
at pH 6.0 and 35 8C (Table 1). These favorable pH and
temperature values are similar to those obtained for suspended
or granular sludge systems inoculated with the same seed
culture [9,15]. Batch tests were also conducted to identify the
best initial sucrose concentration (5–30 g COD l1) when the
most favorable pH (6.0) and temperature (35 8C) were used.
Table 2 shows that both Rmax and Hmax reached the highest
level when the initial sucrose concentration was
20 g COD l1, while both 10 and 20 g COD l1 gave high
Y H2 values of 2.68 and 2.25 mol H2 mol sucrose1, respectively (Table 2). Consistently, our previous free-cell work also
showed that 20 g COD l1 of sucrose resulted in the best H2
producing performance [9,15,16]. The foregoing results
suggest that the cell immobilization procedures did not
considerably alter the H2 production properties of the
bacterial population in the sludge.
3.2. Continuous H2 production using PMMA-immobilized
cells
Using the favorable conditions obtained from batch studies
(i.e., 35 8C, pH 6.0, 20 g COD l1 of sucrose in feed),
continuous cultures were carried out for H2 production under
a progressively decreased HRT (from 8 to 4 h). As indicated in
Fig. 1, the H2 production rate increased when HRT was
decreased from 8 to 6 h, whereas further decrease in HRT to 4 h
resulted in a marked decrease in H2 production rate. Further
decrease in HRT to 2 h resulted in significant washout of
suspended biomass and unstable operation of the system (data
not shown). The best H2 production rate in continuous culture
Fig. 1. Time-course profiles of volumetric H2 production rate and H2 content
performance in continuous culture containing PMMA immobilized cells under
different hydraulic retention time (HRT). (Sucrose concentration in
feed = 20 g COD l1, pH 6.0, temperature = 35 8C.)
was ca. 1.8 l h1 l1 (at HRT = 6 h), which is significantly
higher than the maximum rate obtained from batch cultures
(238 ml h1 l1), indicating a more efficient H2 production
performance in continuous fermentation. Table 3 shows that
operation at HRT = 6 h also gave a slightly better H2 content in
biogas (41%). The H2 yield was similar for HRT = 6 and 8 h
with a value of 1.7–2.0 mol H2 mol sucrose1, but dropped
considerably to 1.1 mol H2 mol sucrose1 at HRT = 4 h
(Table 3). Under identical operation conditions, the continuous
culture with suspended cells (SPC) attained similar H2 yield
and H2 content in biogas to the immobilized cell (IMC) system
at a HRT of 6 and 8 h, while the production rate for SPC was
50–55% lower than that for IMC (Table 3). Meanwhile, the SPC
Table 2
H2 content in biogas and estimated kinetic parameters with modified Gompertz equation (Eq. (1)) in batch fermentation using PMMA immobilized cells under
different initial sucrose concentrations
Sucrose concentration
(g COD l1)
H2 contenta
(%)
H2 production
ratea,b (ml h1 l1)
H2 yielda
(mol H2 mol sucrose1)
Model simulation
Total H2 evolution,
Hmax (ml)
Rmax (ml h1)
l (h)
R2
5
10
20
30
38 1
41 2
43 1
40 0
57.5 6.3
170 7
238 5
67.5 6.1
1.78 0.11
2.68 0.18
2.25 0.11
0.58 0.05
50
150
252
97
5.75
17
23.8
6.75
14.2
13.5
7.8
16.1
0.912
0.973
0.995
0.948
Initial pH 6.0, temperature = 35 8C.
a
The data are the mean values of duplicate tests (the ‘‘’’ denotes standard deviation of duplicates).
b
Maximum volumetric H2 production rate.
282
K.-J Wu, J.-S Chang / Process Biochemistry 42 (2007) 279–284
Table 3
H2 production performance in continuous culture containing PMMA immobilized cells and suspended cells (control) under different hydraulic retention time
(HRT)
HRT
(h)
Culture
type
H2 content
in biogasa (%)
H2 production
ratea (l h1 l1)
H2 yielda
(mol H2 mol sucrose1)
8
IMC
SPC
37 2
38 1
1.42 0.17
0.93 0.07
1.7 .01
1.9 0.3
6
IMC
SPC
41 2
40 1
1.80 0.22
1.21 0.10
2.0 0.2
1.9 0.1
4
IMC
SPC
39 2
wo
0.83 0.10
wo
1.1 0.2
wo
biocatalysts, as the major H2 producers in fixed-bed and CIGSB
system were the surface-attached biofilms [16] and selfflocculated granules [9,12], respectively. In contrast, the H2
producers of this immobilized-cell (IMC) system were
entrapped in the porous polymeric materials. Hence, more
mass transfer limitations may arise from the IMC system than
from biofilm and granular sludge, resulting in the difference in
the effect of HRT on H2 production.
3.3. Carbon substrate utilization and soluble metabolites
production
Sucrose concentration in feed = 20 g COD l1, pH 6.0, temperature = 35 8C.
IMC: immobilized cells, SPC: suspended cells, wo: cell wash-out.
a
The data are the mean values of duplicate tests (the ‘‘’’ denotes standard
deviation of duplicates).
culture was washed out while operating at a HRT of 4 h, at
which the IMC system was able to maintain stable, indicating
that IMC system was more stable against hydraulic dilution
rates. Moreover, compared to our recent continuous H2
producing processes [11,12,16], the present IMC system was
much more stable in terms of H2 production rate (Fig. 1) and
can be stably operated for over 6 months (data not shown). It is
likely that in the immobilized-cell system, most of the bacterial
populations were protected inside the polymeric matrix,
thereby being able to buffer the impact of variations in
environmental factors. However, compared to our recent fixedbed [16] and carrier-induced granular sludge bed (CIGSB) [9]
bioreactors, showing an optimal HRT of 0.5–1 h, the best HRT
in this system was higher (i.e., HRT = 6 h). This difference
could be attributed to different physical characteristics of the
Sucrose conversion in all the batch runs was in general
within 83–95% and was as high as 98–99% in continuous runs,
indicating a good substrate conversion efficiency of the
proposed immobilized-cell system. Accompanying production
of biogas (essentially consisting of 30–40% H2 and 60–70%
CO2) (Tables 1–3), soluble metabolites (e.g., acids and
alcohols) also formed. The major soluble metabolites were
butyric acid (HBu), acetic acid (HAc), and 2,3-butandiol (2,3BuOH). These three products accounted for 81–98% of total
soluble microbial products (SMP) (Table 4). The only
difference compared to our previous H2-producing processes
[6–9,12,13,16] was the formation of a considerable amount of
2,3-BuOH, which was absent in our previous studies. This
result seems to suggest that some facultative anaerobic H2
producers (e.g., Enterobacter or Klebsiella species) could be
present in the immobilized cells, since 2,3-BuOH is one of the
major products of those facultative anaerobes while catabolizing carbohydrates [17]. Indeed, one of the pure strain isolated
from the immobilized-cell beads was identified as Klebsiella
sp. HE1 (NCBI accession no. AY540111) according to 16S
Table 4
Production and composition of soluble metabolites during batch and continuous H2 fermentation with PMMA immobilized cells under different temperature and pH
Operation
mode
Temperature
(8C)
Initial
pH
TVFAa
(mg COD l1)
SMPa
(mg COD l1)
HAc/SMP
(%)
HBu/SMP
(%)
EtOH/SMP
(%)
2,3-BuOH/SMP
(%)
TVFA/SMP
(%)
Batch
30
30
30
30
35
35
35
35
40
40
40
40
5.5
6.0
6.5
7.0
5.5
6.0
6.5
7.0
5.5
6.0
6.5
7.0
763 68
659 75
739 37
919 18
1040 62
1416 42
983 50
1209 108
1023 71
972 49
941 92
855 61
1519 46
1416 113
1391 97
1761 141
1919 96
1808 72
1877 95
1833 110
1590 48
1691 118
1694 85
1630 65
24
20
23
28
24
29
23
28
25
24
25
23
26
27
30
31
31
49
30
38
39
33
31
29
15
16
15
6
13
2
16
7
8
11
17
19
35
37
38
35
32
20
31
27
28
32
27
29
50
47
53
52
54
78
52
66
64
57
56
52
Operation mode
HRT
(h)
TVFAa
(mg COD l1)
SMPa
(mg COD l1)
HAc/SMP
(%)
HBu/SMP
(%)
EtOH/SMP
(%)
2,3-BuOH/SMP
(%)
TVFA/SMP
(%)
Continuousb
8
6
4
920 89
2403 144
1981 60
1545 123
3184 159
3905 234
28
33
22
32
42
29
7
3
9
33
22
40
60
75
51
Initial or feeding sucrose concentration = 20 g COD l1.
a
The data are the mean values of duplicate tests (the ‘‘’’ denotes standard deviation of duplicates).
b
Conducted at pH 6.0 and temperature = 35 8C; HAc: acetic acid; HBu: normal butyric acid; EtOH: ethanol; 2,3-BuOH: 2,3-butandiol; TVFA (total volatile fatty
acid) = HAc + HBu; SMP: soluble microbial products (SMP = TVFA + EtOH + 2,3-BuOH).
K.-J Wu, J.-S Chang / Process Biochemistry 42 (2007) 279–284
rDNA sequence comparison, as the strain has a 99.9% identity
to Klebsiella pneumoniae subsp. ozaenae (NCBI accession no.
AF228919). This finding may explain why 2,3-BuOH was
produced in both batch and continuous cultures. Moreover,
there was a general trend that when the H2 production rate was
higher, the total volatile fatty acid (TVFA) production was
higher, so was the TVFA/SMP ratio (Table 4). This suggests
that acid formation was preferable for H2 production, whereas
production of alcohols was not favorable. These results are
consistent with the common rules that alcohol formation may
consume free electrons carried by metabolic reducing power
(e.g., NADH), thereby inhibiting H2 production [18].
3.4. Novelty, significance, and limitation of the proposed
immobilized-cell system
In our recent work, several cell-entrapment-type immobilized-cell systems were developed [6–8]. However, most of
them (e.g., CA-based and PU immobilized cells [6]) have not
been successfully utilized in continuous fermentation, due
mainly to the insufficient mechanical strength and stability for
long-term operation. That was the reason why a stronger
polymeric matrix (i.e., PMMA) was used in this study to create
a more stable immobilized-cell beads suited for a prolonged
continuous operation. It is not easy to use a rigid polymer, like
PMMA, as a matrix for cell immobilization. The key technique
was the addition of supplemental materials to modify the
properties of the matrix and to create appropriate pore size. To
our best knowledge, this is the first attempt of using PMMA to
immobilize cells for H2 production or other applications.
Indeed, the resulting PMMA-immobilized cells displayed a
very stable performance in continuous production of H2 from
sucrose, allowing stable operation for over 6 months (data not
shown). In addition, the PMMA-immobilized cells were able to
achieve a high H2-prodcution rate at a relatively low dilution
rate (high HRT). Despite operation at a high HRT of 6 h, the
PMMA-immobilized cells attained a H2 production rate of
1.8 l h1 l1, which is higher than most of reported values [1,2]
and similar to the maximal H2 production rate (1.32 l h1 l1)
achieved by our previous fixed-bed process at a much shorter
HRT of 1 h [16]. This special feature clearly suggests that using
PMMA cells might reduce the operational cost by gaining a
comparable H2 producing capacity at a much lower organic
load rate (or a much longer HRT).
Although the H2 yield (up to 2.68 mol H2 mol sucrose1)
obtained from the present study is considerably higher than our
recent immobilized-cell systems [7,8], the yield is still lower
than that obtained from the suspended-cell systems [9,14–16].
The major cause for the lower yield could be due to the presence
of an unfavorable bacterial community structure or the mass
transfer limitations arising from cell entrapment. It is likely that
the lower pH or higher H2 and CO2 concentration may be
present within the entrapped cells due to accumulation of acidic
metabolites and gas products, resulting in earlier termination of
H2 production or unfavorable H2-producing kinetics. Nevertheless, this limitation might be overcome by an appropriate
adjustment of the pore size of the PMMA cells.
283
4. Conclusions
This work demonstrated a feasible immobilized-cell
system for batch and continuous H2 production with a high
stability and efficiency. The cells entrapped with novel
composite polymeric matrix (PMMA/collagen/activated carbon) displayed good mechanical strength and H2-producing
activity. Batch tests were conducted to explore favorable
conditions for H2 production with the PMMA immobilized
cells. The best sucrose concentration, pH, and temperature
obtained from batch fermentation were 20 g COD l1, 6.0,
and 35 8C, respectively, giving a H2 production rate of
238 ml h1 l1 and a yield of 2.25 mol H2 mol sucrose1. In
continuous culture, the best H2 producing performance
occurred when it was carried out at a relatively high HRT
of 6 h, attaining an excellent H2 production rate of
1.8 l h1 l1 and a H2 yield of 2.0 mol H2 mol sucrose1.
The outcome of this work suggests the potential of using this
immobilized-cell system for continuous H2 production in
practice.
Acknowledgements
The authors gratefully acknowledge the financial support of
Taiwan’s National Science Council (Grant Nos. 93-2211-E006-040 and NSC 94-2211-E-006-026) and Taiwan’s Bureau of
Energy (Grant Nos. NSC93-ET-7-006-001-ET and NSC94-ET7-006-004-ET).
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